Phagosomes Acquire Nascent and Recycling Class II MHC Molecules but Primarily Use Nascent Molecules in Phagocytic Antigen Processing

Cells and media

Peritoneal macrophages were harvested 4 days after i.p. inoculation of mice with Con A (Sigma, St. Louis, MO). B6D2F₁/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). H2-DM knockout mice (45) were generously provided by L. Van Kaer (Vanderbilt University, Nashville, TN) and then bred at Case Western Reserve University. The T cell hybridoma DOBW (46), which recognizes OVA_323–339 bound to I-A^d, derives from a transgenic mouse expressing the TCR from the OVA-specific DO11.10 T hybridoma (47) and was produced by Osami Kanagawa (Washington University, St. Louis). Unless otherwise stated, all experimental incubations were conducted at 37°C in 5% CO₂ in standard medium: DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS (HyClone, Logan, UT), 5 × 10⁻⁵ M 2-ME, l-arginine HCl (116 mg/L), l-asparagine (36 mg/L), NaHCO₃ (2 g/L), sodium pyruvate (1 mM), 10 mM HEPES buffer, and antibiotics.

Preparation of Ag-conjugated beads

Latex beads (2 μm, fluorescent or nonfluorescent; Polysciences, Warrington, PA) or magnetic latex beads (1–2 μm; Polysciences) were noncovalently conjugated with Ag by incubation at 4°C for 48 h with OVA (Sigma), hen egg lysozyme (HEL; Sigma), or RNase (Sigma) at 10 mg/ml in citrate buffer, pH 4.2, for OVA or phosphate buffer, pH 7.4, for HEL and RNase. Conjugated beads were washed in conjugation buffer and then washed and resuspended in standard medium (yield, 10¹² beads/ml and 100 μg OVA per ml, 400 μg HEL per ml, or 400 μg RNase per ml). The conjugated beads were stored at 4°C and used within 3 wk.

Antibodies

The following mAbs were prepared as hybridoma supernatants: 1D4B, a rat IgG2a specific for murine lysosome-associated membrane protein (LAMP)-1; 34-5-3S, a murine IgG2a that recognizes both I-A^d and I-A^b; KL295, a murine IgG1 that recognizes β chains of I-A^d and I-A^b. 1D4B was obtained from the Developmental Studies Hybridoma Bank (Johns Hopkins University, Baltimore, MD and University of Iowa, Iowa City, IA). 34-5-3S and KL295 were obtained from American Type Culture Collection (Manassas, VA). Mouse antiserum against OVA was generated by immunization of C57BL/6 mice with whole OVA protein in CFA. Rabbit antiserum specific for the α-chain of H2-DM was generously provided by John Monaco and Helena Russell (University of Cincinnati, Cincinnati, OH).

Ag processing and presentation assays

Macrophages were plated for 2 h in 96-well plates at 2 × 10⁵ cells/well, and nonadherent cells were removed by washing. The macrophages were incubated with or without BFA (1 μg/ml, Sigma) for 0 min, 30 min, or 3 h at 37°C. Latex-conjugated Ag was added to each well in a final volume of 200 μl, and the beads were pelleted onto the cells by centrifugation at 900 × g for 5 min at 37°C. The cells were incubated at 37°C for an additional 5 min (providing a total pulse period of 10 min) and washed in ice-cold DMEM to remove extracellular beads. Prewarmed medium was added, and the cells were incubated at 37°C for 30 min in the continued presence or absence of BFA. The macrophages were fixed with 0.5% paraformaldehyde and washed. T hybridoma cells (1 × 10⁵) were added to each well (200 μl total volume) and incubated for 20–24 h. Supernatants (100 μl) were harvested and assessed for IL-2 content using the CTLL-2 proliferation assay. CTLL-2 proliferation was monitored by the addition of Alamar blue (Alamar Biosciences, Sacramento, CA) as an indicator dye and measured as the difference between absorbance at 550 nm and 595 nm after 24 h (48). Blanks for spectrophotometry were provided by wells containing medium (added at the initiation of CTLL-2 assay) and Alamar blue (added at the same time as for the other wells). All analyses were performed in triplicate.

Isolation of magnetic latex-OVA phagosomes for T cell assays

Macrophages were plated at 10⁷ cells per well in five 6-well plates. The macrophages were incubated with or without BFA (1 μg/ml) for 3 h at 37°C. Magnetic latex-OVA was added (10 μl/well), and the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations (described above). The cells were detached by scraping, resuspended in homogenization buffer (0.25 M sucrose and 10 mM HEPES, pH 7.2), and homogenized in a Dounce homogenizer (Kontes, Vineland, NJ) to obtain complete lysis of the cells (44). Phagosomes containing magnetic latex-OVA beads were removed with a magnetic particle concentrator (Dynal, Great Neck, NY), washed three times in 2 ml homogenization buffer, and resuspended in 150 μl media. Microscopic examination showed that there were no intact cells contaminating the magnetic phagosome preparation. Each fraction was divided into replicate aliquots, freeze-thawed to disrupt phagosomal membranes, and combined with 10⁵ T hybridoma cells in a final volume of 200 μl in 96-well plates. The plates were incubated for 24 h, the supernatants (100 μl) were harvested, and IL-2 content was assessed using the CTLL-2 proliferation assay as described above.

Flow cytometry and analysis of isolated phagosomes by flow organellometry

Macrophages were plated at 10⁷ cells per well in 6-well plates. The macrophages were incubated with or without BFA (1 μg/ml) for 3 h at 37°C. Latex-OVA was added (10 μl/well), and the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations (described above). Fluorescent latex-OVA (i.e., OVA conjugated to fluorescent latex beads, see above) was used for analyses of intact cells containing phagosomes by flow cytometry, and the cells were washed, detached by scraping, and fixed in 1% paraformaldehyde. For analyses of phagosomes by flow organellometry, nonfluorescent latex-OVA was used, and the cells were subsequently detached by scraping, resuspended in homogenization buffer (0.25 M sucrose and 10 mM HEPES, pH 7.2) containing protease inhibitors (1.0 mM PMSF, 1 μg/ml pepstatin A, and 20 μg/ml leupeptin (Sigma)), and homogenized in a Dounce homogenizer (Kontes) to obtain 80–85% lysis (43). Intact cells and nuclei were removed by centrifugation (200 × g, 10 min). The supernatant was centrifuged at 1900 × g for 10 min to pellet the crude phagosome preparation, which was paraformaldehyde-fixed, washed, and resuspended in PBS as described (43). The resulting phagosome titer was ∼10⁷ phagosomes/ml. The crude phagosome preparation was immunolabeled for flow organellometry as described (43) using a three-step staining strategy and buffer containing saponin to allow access to lumenal epitopes.

Sucrose density gradient isolation of phagosomes and Western blotting analysis

Macrophages were plated at 10⁷ cells per well in two 6-well plates and incubated with or without BFA (1 μg/ml) for 3 h at 37°C. Fluorescent latex-OVA was added (10 μl/well), and the plates were processed as described above to achieve 10-min pulse and 30-min chase incubations. A crude phagosome preparation was prepared (above), and phagosomes were purified on a sucrose density gradient as described previously (44). Phagosomes were collected from the interface of the 10 and 21% sucrose solutions, diluted in PBS, and pelleted in 1.5-ml microcentrifuge tubes. The pellets were immediately frozen on dry ice and stored at −80°C. To provide controls for Western blot analyses, macrophages obtained from CBA/J and H2-DM knockout mice were lysed at a concentration of 5 × 10⁷/ml in lysis buffer (PBS containing 1% Nonidet P-40, 5 mM EDTA, 50 mM iodoacetamide, 1 mM PMSF, 2 μg/ml pepstatin, and 20 μg/ml leupeptin (Sigma)). Phagosomes isolated from sucrose density gradients were similarly lysed. Lysates were either boiled or not boiled under reducing conditions in SDS-PAGE sample buffer (12 mM Tris-HCl, pH 6.8, 5% glycerol, 0.4% SDS, 2.88 mM 2-ME, and 0.02% bromophenol blue) and electrophoresed on 12% SDS polyacrylamide gels (49). The proteins were blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA), probed with H2-DM-specific rabbit antiserum, KL295, or 1D4B, incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (Amersham, Arlington Heights, IL; Pierce, Rockford, IL), and detected using an enhanced chemiluminescence (ECL) kit (Amersham).

ECL exposure times were chosen carefully (after analysis of multiple exposure times) to provide results within a linear detection range and allow reliable quantitation. Furthermore, both appropriate ECL exposure time and appropriate sample loading were confirmed experimentally. SDS-PAGE gels were run for Western analysis with varying numbers of phagosomes per lane. Quantitation of MHC-II by Western analysis with ECL detection showed linearity over at least the 8-fold range of loaded phagosome material that was assessed. Assays shown in this report were done with a load of phagosome material that provided a signal at least 2-fold below the upper limit of the linear range and at least 4-fold above the lower limit.

Analysis of the source of MHC-II in phagosomes following BFA treatment of cells and biotinylation of the cell surface

Macrophages were plated at 10⁷ cells/well in 6-well plates and were either left untreated, cell-surface biotinylated, or incubated with BFA (1 μg/ml for 3 h at 37°C) and then cell-surface biotinylated. For cell-surface biotinylation, cells were incubated with sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS, pH 8.0, for 4 h at 4°C and then washed three times with cold PBS, pH 8.0. Fluorescent latex-OVA was added (10 μl/well), the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations, and phagosomes were purified on a sucrose density gradient and lysed in 0.1 ml lysis buffer as described above. Latex beads were removed by centrifugation, and lysates were divided into two equal aliquots. One aliquot was stored at −80°C, and the other aliquot was incubated on ice with occasional mixing for 1 h with 50 μl pelleted streptavidin magnetic beads (Sigma) to deplete biotinylated proteins. Streptavidin magnetic beads were removed with a magnetic particle concentrator (Dynal), and the entire depletion step was repeated once more to ensure complete removal of all biotinylated proteins. Lysates were then either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions, electrophoresed on 12% SDS polyacrylamide gels (49), transferred onto a polyvinylidene difluoride membrane, and analyzed by Western blot as described above.

Phagocytic processing of latex-OVA is inhibited by BFA

We have previously shown that peritoneal macrophages from B6D2 mice rapidly process latex-OVA and present OVA_323–339:I-A^d complexes to DOBW T hybridoma cells (44). Complexes were found to appear on the cell surface as early as 10 min, and further delivery of additional complexes to the cell surface occurred for 1–4 h. Furthermore, OVA_323–339:I-A^d complexes were shown to form directly within phagosomes that contained the antigenic particles and were not found in endocytic compartments (e.g., MIIC) or other phagosomes within the same cells that contained different particles of unrelated antigenicity (44). These results demonstrated that phagosomes are fully functional Ag-processing organelles that directly mediate the formation of peptide:MHC-II complexes, but the source of MHC-II molecules used in phagocytic processing remained unclear. In the studies presented here, biochemical means were used to analyze I-A molecules (both I-A^d and I-A^b) in phagosomes from macrophages of B6D2F₁ (H-2^{b × d}) mice. In addition, T cell assays were used to detect peptide-MHC-II complexes formed in the course of phagocytic Ag processing by macrophages from inbred mice of several different MHC haplotypes.

To evaluate whether phagocytic Ag processing and presentation used preexisting (i.e., recycling) or nascent MHC-II, we determined the effect of BFA on phagosome composition and the ability of B6D2 macrophages to process OVA to form OVA_323–339:I-A^d complexes. Prior studies have established that incubation with BFA for 3 h depletes the reservoir of nascent MHC-II within the endocytic pathway of macrophages that is required for processing of many epitopes within late endocytic compartments (32, 36). In contrast, treatment with BFA for 30 min blocks transport of nascent MHC-II from the TGN but does not fully deplete nascent MHC-II within endocytic compartments. Accordingly, macrophages were first incubated with or without BFA for 0.5 or 3 h. In the continued absence or presence of BFA, the cells were then incubated with latex-OVA for 10 min, washed, and incubated for 30 min to allow complete internalization of latex-OVA into phagosomes (44). The cells were then either fixed, washed, and incubated with DOBW T hybridoma cells to assess the presentation of OVA_323–339:I-A^d complexes or subjected to subcellular fractionation for analysis of phagosomes.

A substantial inhibition of macrophage OVA processing (approximately a 1 log shift in the dose-response curve) was consistently observed when BFA was added 3 h before the addition of latex-OVA (Fig. 1⇓A). BFA similarly inhibited processing of latex-HEL (Fig. 1⇓C) and latex-RNase (Fig. 1⇓D) by CBA/J macrophages for presentation to 3A9 and TS12 hybridoma cells, respectively. Thus, studies with three different Ag epitopes indicated inhibition of processing by BFA, although future studies are desirable to assess additional epitopes to determine any potential differences in processing mechanisms.

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FIGURE 1.

BFA inhibits processing and presentation of latex-conjugated Ags by macrophages. Macrophages were treated with BFA for 0 min, 30 min, or 3 h and were either fixed immediately for subsequent presentation of exogenous peptide or pulsed with Ag-conjugated latex beads for 10 min, chased for 30 min at 37°C in the continued presence or absence of BFA, and then fixed. T hybridoma cells were added to detect presentation of peptide:MHC-II complexes, and supernatants from T hybridoma assays were assessed for IL-2 with a CTLL-2 proliferation assay monitored by Alamar blue, an indicator dye (see Materials and Methods). Data points are means of triplicate samples ± SD. A, Processing and presentation of latex-OVA by B6D2 macrophages. B, Presentation of exogenous OVA_323–339 peptide by fixed B6D2 macrophages. C, Processing and presentation of latex-HEL by CBA/J macrophages. D, Processing and presentation of latex-RNase by CBA/J macrophages.

The BFA-induced inhibition of Ag processing was not a consequence of reduced uptake of particulate Ag, because BFA did not affect the average number of latex-bead phagosomes formed per macrophage (17 phagosomes/cell after 10-min pulse, 30-min chase, data not shown, determined by flow cytometry as previously described (43)). These results suggest that nascent MHC-II was required for most Ag-processing activity. Recycling MHC-II may have contributed to the minor residual processing activity that persisted at similar levels after treatment with BFA for 2–4 h (data not shown). Incubation with BFA (3 h) had no effect on the ability of subsequently fixed macrophages to present synthetic OVA_323–339 peptide (Fig. 1⇑B), RNase_42–56 peptide (data not shown), or HEL_48–61 peptide (data not shown), indicating that cell-surface expression of MHC-II was not substantially reduced by this treatment. However, when BFA was added only 30 min before latex-OVA, processing was not substantially inhibited (Fig. 1⇑A), indicating that processing continued after cessation of MHC-II transport directly from the TGN. This suggests that some phagosomal MHC-II molecules were derived from the post-Golgi reservoir of nascent MHC-II in endocytic compartments. In conclusion, phagocytic Ag processing appears to primarily use nascent MHC-II that is acquired by phagosomes from the reservoir of these molecules that exists within the endocytic pathway, and additional experiments were performed to test this model.

BFA inhibits the formation of OVA_323–339:I-A^d complexes in phagosomes

The hypothesis developed above indicates that inhibition of phagocytic Ag processing by BFA (3 h) was caused by depletion of nascent MHC-II in phagosomal compartments and a decrease in the formation of peptide:MHC-II complexes in phagosomes. Alternatively, peptide:MHC-II complexes may still have been formed after treatment with BFA but were unable to exit from phagosomes to the plasma membrane. To distinguish between these two possibilities, we directly examined the levels of peptide:MHC-II complexes in phagosomes that were isolated by subcellular fractionation of macrophages with or without BFA.

Macrophages were incubated with or without BFA for 3 h, exposed to magnetic latex-OVA under the conditions described above, and homogenized to release intact phagosomes (43, 44). Phagosomes were then isolated using a magnetic concentrator, transferred into 96-well plates, freeze-thawed to disrupt and open phagosomal membranes, and incubated with DOBW T hybridoma cells for 24 h (44). T cell detection of OVA_323–339:I-A^d complexes in phagosomes was monitored by IL-2 secretion. Phagosomes isolated from BFA-treated cells produced substantially lower DOBW responses (reduced by 93–99%) than phagosomes isolated from untreated cells (Fig. 2⇓). This observation confirmed that BFA inhibited the formation of peptide:MHC-II complexes in phagosomes during phagocytic Ag processing.

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FIGURE 2.

BFA inhibits the formation of OVA_323–339:I-A^d complexes in phagosomes. Macrophages were treated with BFA for 0 h or 3 h, pulsed with magnetic latex-OVA beads for 10 min, and chased for 30 min at 37°C in the continued presence or absence of BFA. Following homogenization of the cells, magnetic latex-bead phagosomes were isolated by the application of a magnetic field, aliquots from each preparation were freeze-thawed, and DOBW cells were added to detect OVA_323–339:I-A^d complexes. IL-2 secretion by DOBW cells was assessed as in Fig. 1⇑. Data points are the mean of duplicate samples.

Effect of BFA on expression of MHC-II in phagosomes and phagosomal Ag degradation as determined by flow organellometry

The ability of BFA to inhibit the formation of OVA_323–339:I-A^d complexes in phagosomes could result from decreased levels of phagosomal MHC-II or decreased Ag degradation to produce immunogenic peptides. Previous flow organellometry analysis showed that phagosomes contain MHC-II, acquire lysosomal markers, and degrade particle-associated Ag (43, 44). For example, LAMP-1, a lysosomal membrane marker, is acquired by phagosomes following their fusion with lysosomes. To investigate phagocytic Ag-processing mechanisms and their inhibition by BFA, macrophages were incubated with or without BFA for 30 min or 3 h, exposed to latex-OVA for 10-min pulse and 30-min chase incubations, and homogenized to prepare phagosomes. Phagosomes were fixed in 1% paraformaldehyde and immunolabeled in buffer containing 0.1% saponin to allow Abs access to epitopes in the lumen of phagosomes for detection of phagosomal membrane proteins and components of the phagocytosed particle. Flow organellometry (43, 44) was then used to analytically isolate latex-bead phagosomes from other organelles and cell fragments by gating (due to the unique scatter properties of latex bead phagosomes), determine phagosomal levels of MHC-II and LAMP-1, and monitor the degradation of OVA coupled to latex beads.

Treatment of macrophages with BFA for 3 h caused a substantial reduction in phagosomal MHC-II (Fig. 3⇓). Three independent experiments showed that treatment with BFA decreased the mean fluorescence value (MFV) for staining for I-A^d and I-A^b by 69 ± 4%. In contrast, this treatment caused only a slight reduction in phagosomal LAMP-1 (13 ± 2% decrease in MFV, n = 3). These results indicate that the post-Golgi pool of MHC-II that could access phagosomes was diminished after 3 h of BFA treatment. This suggests that this pool of MHC-II was characterized by a short half-life and was normally dependent on replenishment with nascent MHC-II over a 3-h time span. In contrast, the post-Golgi pool of LAMP-1 that contributes to phagosomal delivery was less affected by treatment with BFA for 3 h, suggesting that the post-Golgi pool of LAMP-1 was characterized by a longer half-life and less dependence on nascent molecules. This also suggests that the post-Golgi compartment in which LAMP-1 resides still exists and can provide delivery of LAMP-1 after a 3-h treatment with BFA.

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FIGURE 3.

Analysis of phagosomal proteins by flow organellometry. Macrophages were treated with or without BFA for 3 h, pulsed with latex-OVA for 10 min, and chased for 30 min in the continued presence or absence of BFA. Phagosomes were prepared, fixed with paraformaldehyde, permeabilized with saponin, stained for LAMP-1, MHC-II, or OVA, and analyzed by flow organellometry. Gating by optical scatter parameters was used to select single-bead phagosomes. A–F, LAMP-1 and MHC-II were detected with the indicated monoclonal Abs. Negative-control staining with isotype-matched control Abs was used to define the H1 gate (negative events). The H2 gate represents the positive events, and the H3 gate represents all the events gated as single-bead phagosomes. G–I, Phagosomes and latex-OVA beads were stained with OVA-specific mouse serum (raised against whole OVA protein, solid lines) or control mouse serum (dashed lines). G, Staining of latex-OVA (not exposed to cells). H and I, Staining of phagosomes containing latex-OVA. The H3 gate represents all single-bead phagosomes.

Incubation of cells with BFA for only 30 min had no effect on the levels of either MHC-II or LAMP-1 in phagosomes (data not shown) or whole-cell processing and presentation of latex-OVA (Fig. 1⇑A). Because BFA rapidly blocks endoplasmic reticulum (ER)/Golgi transport and exit from the TGN, the observation that a 30-min treatment with BFA did not affect phagosome acquisition of MHC-II suggests that MHC-II molecules are not transported directly from the TGN to phagosomes. Rather, MHC-II molecules must reside in an intermediate compartment after exit from the TGN and before phagosomal delivery, and MHC-II molecules apparently linger in this compartment for periods substantially longer than 30 min. All of these findings are consistent with the hypothesis that MHC-II molecules can traffic from the significant reservoir of MHC-II molecules in endosomal compartments to phagosomes.

Flow organellometry was also used to measure the amount of OVA remaining in latex-OVA phagosomes as a measure of Ag degradation and the delivery of proteases to phagosomes. Our previous studies demonstrated the detection of latex bead-conjugated Ag within phagosomes by flow organellometry and revealed that Ag was progressively lost with phagosome maturation (43, 44). In the present studies, phagosomes were similarly labeled with OVA-specific mouse antiserum (raised against whole OVA) and analyzed by flow organellometry. Incubation with BFA starting 30 min or 3 h before the addition of Ag caused little or no change in phagosomal degradation of OVA, and 84% or more of the OVA that was initially associated with latex beads was degraded within 30 min with or without BFA (Fig. 3⇑). Thus, phagosomes acquired proteases from a post-Golgi pool that was not depleted after 3 h of treatment with BFA, consistent with acquisition of proteases from endosomal compartments that remain intact and capable of delivery to phagosomes after treatment with BFA for 3 h.

In summary, treatment of macrophages with BFA for 3 h significantly decreased phagosomal levels of MHC-II but had little effect on phagosome acquisition of lysosomal markers and degradation of phagosomal contents. While all of these components appear to be delivered from endosomes to phagosomes, the putative endosomal source of MHC-II was most dependent on the BFA-sensitive supply of nascent molecules. Additional approaches were developed to detect phagosomal MHC-II molecules by Western analysis and determine the relative contributions of plasma membrane and endosomal pools of MHC-II to phagosomes.

Effect of BFA on levels of H2-DM and MHC-II in phagosomes as determined by Western blotting

H2-DM is an important component in most MHC-II Ag-processing mechanisms, and experiments were performed to determine whether BFA-mediated inhibition of phagosomal Ag processing correlated with any change in expression of H2-DM in phagosomes. Macrophages were incubated with or without BFA for 3 h before phagocytic challenge. Because antiserum to H2-DM was not effective for immunolabeling and flow organellometry, highly purified phagosomes were isolated by sucrose gradient fractionation (44) and subjected to Western blot analysis. In three independent experiments, densitometry of H2-DM bands showed that BFA decreased phagosomal H2-DM levels by only 3–5% (e.g., Fig. 4⇓A, lanes 3 and 5). Thus, treatment with BFA for 3 h had minimal effect on the level of H2-DM in phagosomes. This suggests that H2-DM resides in a vesicular compartment that remains intact and able to deliver H2-DM to phagosomes even after treatment with BFA for 3 h.

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FIGURE 4.

Western blot analysis of phagosomal proteins. A crude preparation of phagosomes was generated as in Fig. 3⇑, and phagosomes were then purified on sucrose gradients. Diluted aliquots of the phagosomes were counted in a hemacytometer to insure that an equal number of phagosomes was used for each Western blot analysis. Purified phagosomes or control samples of whole cells were solubilized in SDS-PAGE sample buffer under reducing conditions and subjected to SDS-PAGE and Western blotting. Samples 1, 3, 5, and 6 were boiled (“B”) before loading, while samples 2 and 4 were not boiled (“NB”). Boiling of the samples dissociates all MHC-II molecules into separate α- and β-chains; without boiling, some mature MHC-II molecules (stabilized by antigenic peptides) remain associated as αβ dimers (49 ). Lysates of macrophages from H2-DM knockout mice and CBA/J mice were used as controls. A, Detection of H2-DM with antiserum specific for the α-chain of H2-DM. B, Detection of the β-chain of 1-A^b or I-A^d with KL295. C, Detection of LAMP-1 with 1D4B.

For analysis of MHC-II, the blots were probed with KL295, an Ab specific for the β-chain of I-A^b or I-A^d. Before electrophoresis, samples were either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions to determine the effect of BFA on the levels of mature (SDS-stable) MHC-II dimers and immature (SDS-unstable) MHC-II dimers (see legend to Fig. 4⇑ and Ref. 49). In seven independent experiments, BFA reduced SDS-stable dimer in unboiled samples (Fig. 4⇑B, lanes 2 and 4), β-chain monomer in unboiled samples (Fig. 4⇑B, lanes 2 and 4), and β-chain monomer in boiled samples (Fig. 4⇑B, lanes 3 and 5). Because phagosomes from cells of B6D2F₁ mice express both I-A^b and I-A^d, it is noteworthy that mature I-A^b molecules (containing high-affinity peptides and not CLIP) are SDS-stable at room temperature, whereas I-A^d molecules dissociate into monomers in SDS regardless of the presence or absence of antigenic peptide (50). Thus, mature MHC-II molecules are not all SDS-stable in this analysis. The most direct measurement of total phagosomal I-A (both I-A^b and I-A^d) was provided by densitometry of KL295 labeling for β-chain monomer in boiled samples. This analysis showed that treatment with BFA for 3 h reduced phagosomal MHC-II by 57 ± 10% (n = 7), only slightly less than the 69% decrease determined by flow organellometry analysis. BFA had little effect on the levels of LAMP-1 in isolated phagosomes (Fig. 4⇑C), consistent with the flow organellometry analysis.

Phagosomes acquire preexisting, BFA-resistant MHC-II from the plasma membrane in addition to nascent MHC-II

The experiments described above indicated that phagosomes acquired nascent MHC-II, which was depleted by prior addition of BFA, but the source of the remaining, BFA-resistant phagosomal MHC-II remained unknown. Because most MHC-II molecules reside on the cell surface and may be internalized by phagocytosis (or endocytosis), the plasma membrane was a potential source of phagosomal MHC-II. Therefore, we investigated the internalization of biotinylated cell-surface MHC-II into phagosomes. In addition, BFA was used to deplete phagosomes of nascent MHC-II to determine the relative contributions of nascent vs preexisting MHC-II and plasma membrane-derived vs intracellularly derived MHC-II to the total level of phagosomal MHC-II.

To follow the trafficking of plasma membrane-derived MHC-II molecules, cells were treated with or without BFA for 3 h, cooled on ice, and surface biotinylated using a membrane-impermeable biotinylation reagent at 4°C for 4 h (see Materials and Methods). Under these conditions, >98% of the surface MHC-II molecules were biotinylated and could be removed with streptavidin before detection of MHC-II by Western blotting (data not shown). The cells were then subjected to a 10-min pulse incubation with latex-OVA and a 30-min chase incubation in the continued presence or absence of BFA. Phagosomes were isolated on sucrose density gradients, lysed, and divided into two equal aliquots. Biotinylated proteins were depleted from one of the aliquots by two cycles of incubation with streptavidin-magnetic beads. Phagosomal lysates were either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions before electrophoresis and transfer. The blots were probed with streptavidin-HRP to confirm that all biotinylated proteins had indeed been removed in the depleted samples (data not shown). Phagosomal MHC-II and LAMP-1 were detected by Western blot analysis (Fig. 5⇓).

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FIGURE 5.

Phagosomes acquire surface MHC-II in addition to nascent molecules. Macrophages were left untreated, cell-surface biotinylated, or treated with BFA (3 h) followed by cell-surface biotinylation. Latex-OVA was added, and the cells were incubated in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations. Latex-OVA phagosomes were purified on sucrose gradients, solubilized, and divided into two equal aliquots. From one aliquot, all biotinylated proteins were depleted by two cycles of addition of streptavidin magnetic beads. The samples were then either not boiled (A) or boiled (B and C), subjected to SDS-PAGE, blotted onto membranes, and probed as in Fig. 4⇑. A and B, Detection of the β-chain of 1-A^b or I-A^d with KL295. C, Detection of LAMP-1 with 1D4B.

Streptavidin-depletion of biotinylated molecules from phagosomal samples resulted in substantial loss of MHC-II. In particular, streptavidin-depletion decreased β monomer in boiled samples (e.g., Fig. 5⇑B, lanes 3 and 4), dimer in unboiled samples (e.g., Fig. 5⇑A, lanes 3 and 4), and β monomer in unboiled samples (e.g., Fig. 5⇑A, lanes 3 and 4). In unboiled samples, the dimer band was more sensitive to streptavidin depletion than the β monomer band. This suggested that the proportion of MHC-II molecules that were SDS-stable dimers was higher in surface-derived MHC-II than intracellularly derived MHC-II (which presumably included a higher proportion of nascent MHC-II).

To determine the relative contributions of different MHC-II source pools to phagosomes, densitometry was performed on Western blots of phagosomal samples from two independent experiments to assess the level of I-A β-chain (results shown in Table I⇓, refer to lanes 3–6 in Fig. 5⇑B). In Fig. 5⇑B, lane 3 shows total phagosomal I-A from surface-biotinylated cells, lane 4 shows intracellularly derived (streptavidin-resistant) I-A, lane 5 shows preexisting (BFA-resistant) I-A, and lane 6 shows preexisting intracellularly derived I-A molecules. The percent contribution of each source of phagosomal I-A was determined by dividing the densitometry value for the source band (e.g., preexisting I-A, lane 5) by the value derived for total phagosomal I-A (lane 3). The contribution of other subsets of I-A source pools, e.g., preexisting or surface-derived, was calculated from the preceding data (see Table I⇓). In these experiments, BFA analysis indicated that 33 ± 6% of phagosomal I-A was derived from preexisting molecules, and 66 ± 6% was derived from the nascent pool. The streptavidin-depletion studies indicated that 60 ± 3% of phagosomal I-A was intracellularly derived, and 40 ± 3% was cell-surface derived. Of the preexisting I-A molecules, most (22 ± 4% of total I-A) were surface-derived, and a minority (11 ± 2% of total I-A) were intracellularly derived (presumably reflecting preexisting I-A molecules that were in endocytic compartments when the cells were biotinylated). Of nascent I-A, most (49 ± 5% of total I-A) were derived from intracellular sources (possibly MIIC or other endocytic compartments), and a minority (18 ± 1% of total I-A) was derived from the cell surface. In interpreting these numbers, it is important to realize that our functional definition of nascent MHC-II includes all molecules synthesized within 3 h, regardless of whether some of these molecules may have been loaded with antigenic peptide and exported to the cell surface within this time frame. In summary, nascent MHC-II comprised 66% of phagosomal MHC-II, mostly delivered from intracellular compartments, whereas preexisting MHC-II accounted for 33% of phagosomal MHC-II and was mostly derived from the cell surface. Fig. 6⇓ presents a model of the major immediate sources of phagosomal MHC-II molecules.

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FIGURE 6.

Sources of phagosomal MHC-II molecules. This model distinguishes different immediate sources of phagosomal MHC-II (e.g., endocytic compartments vs plasma membrane, newly synthesized vs preexisting) but does not show all potential prior stages of MHC-II transport. For example, pathway “H” may involve transit of MHC-II through endocytic compartments (not shown) before its appearance on the cell surface. Letter designations next to pathways refer to row numbers in Table I⇓.